Patent GB-811757-A (GB)
Title: Production of an antibiotic designated antibiotic 101a fermentation
Filed: 1956-10-23
Abstract: An antibiotic designated antibiotic 101a is produced by cultivating Streptomyces spectabilis NRRL 2494 novspec, or a variant or mutant thereof, in an aqueous nutrient medium containing assimilable carbohydrate and proteinaceous material under aerobic and preferably submerged aerobic conditions.  An initial pH of 6.5 to 7.5, a temperature of cultivation of 24 DEG  to 37 DEG  C. and preferably 28 DEG  to 34 DEG  C. and a duration of cultivation of 2 to 6 days is employed.  Specified sources of (a) carbohydrate are glucose, dextrin, molasses, starch, maltose, galactose, mannitol and soybean oil; (b) nitrogen are soybean meal, fish meal, cotton seed meal, defatted soybean meal, peanut meal, brewers' yeast, corn gluten and corn steep <PICT:0811757/IV (b)/1> <PICT:0811757/IV (b)/2> extraction or (b) adsorption on carbon followed by elution.  The broth is filtered at pH 4 to 8, extracted with a water-immiscible organic solvent, the extract concentrated to a solid which is redissolved in a solvent and 101a is precipitated by the addition of a non-solvent, e.g. n-hexane.  Typical solvents are alkyl acetates, e.g. ethyl acetate and amyl acetate, aliphatic ketones such as methyl ethyl ketone, halogenated aliphatic hydrocarbons such as methylene chloride and hydrocarbons such as benzene.  The antibiotic 101a isolated from most beers contains five antibiotically active components designated 101a-1, 101a-2, 101a-3, 101a-4 and 101a-5.  Additional components 101a-6 and 101a-7 have been observed in some beers.  In benzene-methanol-water, the Rf values of these components are 0.65, 0.35, 0.75, 0.85 (4 and 5 together), 0.25 and 0.55 respectively.  The components are partially separated by (a) partition chromatography and (b) Craig countercurrent extraction, a combination of (a) and (b) being necessary to obtain complete separation.  A water-ethanol-cyclohexane-ethyl acetate (1 : 1 : 1 : 1) solvent system may be used in the Craig extraction process. Component 101a-2 is converted to the other components by treatment liquor.  Other additions are (a) inorganic salts, e.g. such as produce ions of potassium, sodium, calcium, phosphate and sulphur; (b) trace elements.  Antibiotic 101a is recovered by (a) with (a) acetic anhydride, (b) p-toluene sulphonate and glacial acetic acid, and (c) ammonia and ethanol.  In the case of (b), component 101a-1 only is produced.  Crystalline 101a samples melt at 155 DEG  to 255 DEG  C.; show ultraviolet absorption maxima at 435 mm  and 245 mm ; contain 63.64 per cent carbon, 6.89 per cent hydrogen and 1.68 per cent nitrogen, the balance being oxygen; soluble in alcohols such as methanol, alkyl acetates such as amyl acetate, alkyl ketones such as methyl ethyl ketone, chlorinated aliphatic hydrocarbons such as methylene chloride, dioxane; substantially insoluble in n-hexane, water and ethers; unstable in alkali, stable at pH 2 to 6; infra-red absorption spectrum as in Fig. 2; neutral or weakly acidic hydrolysing on treatment with dilute alkali; forms helianthate with alcoholic solution of methyl orange; forms acyl derivatives with acylating agents, e.g. acetyl and benzoyl derivatives; active against a wide range of micro-organisms including pneumococci, staphylococci and M. tuberculosis.  Antibiotic 101a and its components are used in formulations for the treatment of man and animals (see Group VI). The characteristics of components 101a-1 to 101a-7 are as follows: <TABS:0811757/IV (b)/1> Vitamin B12 is produced by growing Streptomyces spectabilis NRRL 2494 in a nutrient medium containing cobalt ions.  A steroid oxidized in the 11 position is obtained using S. spectabilis in the presence of a steroid precursor such as progesterone or Reichstein's compound S. or S. acetate.ALSO:Antibiotic 101a, or a component thereof (i.e. antibiotics 101a-1, 101a-2, 101a-3, 101a-4, 101a-5, 101a-6, 101a-7), produced by Streptomyces spectabilis NRRL 2494 nov/spec. (see Group IV(b)), alone or in admixture with other antibiotics or therapeutic agents is employed as a feed supplement in promoting the growth of animals and poultry.ALSO:Antibiotic 101a, or a component thereof (i.e. antibiotics 101a-1, 101a-2, 101a-3, 101a-4, 101a-5, 101a-6 or 101a-7), produced by culturing Streptomyces spectabilis NRRL 2494 Nov. spec. (see Group IV (b)), as such or in admixture with a pharmaceutical carrier is used in the treatment of microbial infections especially staphylococcal, streptococcal and tubercular diseases of man and animals.  Suitable formulations are tablets, capsules, powders, granules, aqueous solutions or suspensions and suspensions in edible oils for oral or parenteral use.  The suspensions may contain a suspending agent such as gelatin, tragacanth, methyl cellulose, sodium carboxymethyl cellulose and polyvinyl pyrrolidone.  Suspensions in an edible oil such as coconut or peanut oil may contain aluminium, monostearate. Topical preparations include ointments made with petroleum-type grease bases or water-soluble polyethylene glycol bases; creams, emulsions, lotions, sprays and suppositories.  For veterinary use, the preparations are used in the form of bougies, capsules, tablets, ointments and oil suspensions.  Antibiotic 101a may be used in admixture with (a) antitubercular drugs such as isonicotinic acid hydrazide, streptomycin and/or dihydrostreptomycin, p-aminosalicylic acid and salts thereof, cycloserine; a combination of isonicotinic acid hydrazide and p-aminosalicylic acid and salts thereof; and a combination of streptomycin, isonicotinic acid hydrazide and p-aminosalicylic acid and the salts thereof, (b) sulpha drugs such as sulphadiazine, sulphamerazine and sulphamethazine, (c) other antibiotics such as tetracycline, oxytetracycline, chlortetracycline, neomycin, polymyxin, chloramphenicol, penicillins G, O and V, novobiocin, bacitracin, streptothricin, circulin and erythromycin, (d) vitamins such as thiamin, riboflavin, ascorbic acid, niocinamide, pyridoxine, pantothenic acid, Vitamin B12 and folic acid, (e) corticoids such as cortisone, hydracortisone, and esters thereof; D 1-cortisone and D 1-hydrocortisone and their 21-position esters, e.g. acetate, cyclopentylpropionate and succinate; alkyl substituted cortisones and hydrocortisones such as 2-methylhydrocortisone and 6-methylhydrocortisone and esters thereof, (f) other drugs. Antibiotic 101a may be mixed with animal (including poultry) feed compositions for the treatment of disease.